Co-administration of a thrombolytic and an anti-CD18 antibody in stroke

ABSTRACT

A method for improving clinical outcome in focal ischemic stroke in a mammal by increasing cerebral blood flow and/or reducing infarct size is described which involves administering an effective amount of an anti-CD18 antibody to the mammal, in the absence of removal of the arterial obstruction.

RELATED APPLICATION

[0001] This application is a continuation of application Ser. No.09/811,384, filed Dec. 20, 2000, claiming priority under 35 U.S.C. § 120to non-provisional application U.S. Ser. No. 09/251,652, filed Feb. 17,1999, which is a continuation-in-part application claiming priorityunder 35 U.S.C. § 120 to non-provisional application U.S. Ser. No.08/788,800, filed Jan. 22, 1997, which claims priority under 35 U.S.C. §119(e) to provisional application U.S. Serial No. 60/093,038, filed Jan.23, 1996, (which was converted from non-provisional application U.S.Ser. No. 08/589,982 by petition), the entire disclosures of whichnon-provisional and provisional applications are incorporated herein byreference.

[0002] This invention was made with United States government supportunder grant NS31008 and NS28708 awarded by the National Institutes ofHealth. The United States government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] This invention relates generally to the use of anti-CD18antibodies for treating stroke. In particular, it relates to the use ofanti-CD18 antibodies for improving clinical outcome by increasingcerebral blood flow and/or reducing infarct size in focal ischemicstroke caused by obstruction of a main cerebral artery.

[0005] 2. Description of Related Art

[0006] Stroke is a general term for acute brain damage resulting fromdisease of blood vessels. This presents a serious problem to society,with about 500,000 people dying from or becoming permanently disabled bystroke in the United States each year. Stroke can be classified into twomain categories: hemorrhagic stroke (resulting from leakage of bloodoutside of the normal blood vessels) and ischemic stroke (cerebralischemia due to lack of blood supply); this application is primarilyconcerned with the latter.

[0007] The three main mechanisms of ischemic stroke are thrombosis,embolism and systemic hypoperfusion (with resultant ischemia andhypoxia). In each of these types of stroke, the area of the brain thatdies as a result of the lack of blood supply thereto is called aninfarct. Obstruction of a cerebral artery resulting from a thrombuswhich has built up on the wall of a brain artery is generally calledcerebral thrombosis. In cerebral embolism, the occlusive materialblocking the cerebral artery arises downstream in the circulation (e.g.an embolus is carried to the cerebral artery from the heart). Because itis difficult to discern whether a stroke is caused by thrombosis orembolism, the term thromboembolism is used to cover both these types ofstroke. Systemic hypoperfusion may arise as a consequence of decreasedblood levels, reduced hematocrit, low blood pressure or inability of theheart to pump blood adequately.

[0008] When symptoms of stroke last less than 24 hours and the patientrecovers completely, the patient is said to have undergone a transientischemic attack (TIA). The symptoms of TIA are a temporary impairment ofspeech, vision, sensation or movement. Because a TIA is often thought tobe a prelude to full-scale stroke, patients having suffered a TIA arecandidates for prophylactic stroke therapy with anticoagulation agents(e.g., coumarin and heparin) or antiplatelet agents (such as aspirin andticlopidine) for example.

[0009] Thrombolytic agents, such as tissue plasminogen activator (t-PA),have been used in the treatment of thromboembolic stroke. Thesemolecules function by lysing the thrombus causing the ischemia. Suchdrugs are believed to be most useful if administered as soon as possibleafter acute stroke (preferably within 3 hours) in order to at leastpartially restore cerebral blood flow in the ischemic region and tosustain neuronal viability. In that such drugs exacerbate bleeding,their use in hemorrhagic stroke is contra-indicated.

[0010] A family of adhesion glycoproteins present on leukocytes iscalled the integrin family. This integrin family includes LFA-1(CD11a/CD18), Mac-1 (CD11b/CD18) and p150,95 (CD11c/CD18). Afurthermember of this family CD11d/CD18 has recently been reported. Danilenkoet al., J. Immunol. 155:35-44 (1995). Each of these heterodimers has aunique a-chain (CD11a, b, c or d) and the invariant β-chain (CD18). CD18integrins located on leukocytes may bind to intercellular adhesionmolecule-1 (ICAM-1) which is expressed on vascular endothelium and othercells, thereby mediating leukocyte adhesion and transendothelialmigration.

[0011] It has been noted that CD11 a and CD18 are upregulated inleukocytes from patients who have undergone ischemic stroke or a TIA.Kim et al., J. Neurolog. Sci. 128(1):45-50 (1995). Schroeter et al., J.Neuroimmunology 55(2):195-203 (1994) found that increased expression ofICAM-1 on vessels and leukocytes occurred following cerebral ischemiainduced by permanent occlusion of the middle cerebral artery (MCA) inthe rat.

[0012] The role of cell adhesion molecules in brain injury followingtransient MCA occlusion in the rat has been studied (Matsuo et al.,Brain Research 656:344-352 (1994)). Matsuo et al. inserted a nylonthread from the lumen of the external carotid artery (ECA) to that ofthe right internal carotid artery (ICA) in order to occlude the originof the right MCA. The occlusion was transient; after 1 hour, the nylonthread was removed to allow complete reperfusion of the ischemic areavia the right common carotid artery (CCA). Anti-CD11a (WT1), anti-CD18(WT3) and anti-ICAM-1 (1A29) antibodies were administered beforeischemia and immediately after reperfusion. These researchers found thattreatment with individual antibodies against cell adhesion moleculesreduced edema formation, infarct size and neutrophil accumulationfollowing reperfusion.

[0013] Others have investigated the effects of antibodies against celladhesion molecules in such transient stroke models. Zhang et al., BrainResearch 698:79-85 (1995) studied the effects of anti-CD11b andanti-CD18 monoclonal antibodies in ischemia/reperfusion injury, whereinthe antibodies were administered upon and after transient MCA occlusion(the origin of the MCA was transiently blocked with a surgical nylonfilament). Mori et al., Stroke 23(5): 712-718 (1992) studied the effectsof the anti-CD18 IB4 antibody in their baboon model of reversible MCAocclusion. In this model, arterial obstruction was achieved by inflatingan extrinsic MCA balloon to 100 μl. Reperfusion occurred followingballoon deflation. See, also, Chopp et al., Stroke 25(4):869-876 (1994)and Chen et al., Annals of Neurology35(4): 458-463 (1994) concerning ananti-CD11b antibody in a transient cerebral ischemia model. Chopp etal., and Chen et al., advanced a surgical nylon suture into the ICA toblock the origin of the MCA. Reperfusion was accomplished by withdrawingthe suture until the tip cleared the ICA lumen.

[0014] Takeshima et al., Stroke, 23(2):247-252 (1992) found that theanti-CD18 antibody 60.3 did not afford protection from severe focalischemia and reperfusion in a transient focal cerebral ischemia model incats. Takeshima et al. used a microvascular clip to occlude the MCA andoccluded CCAs by tightening previously placed ligatures.

[0015] Lindsberg et al. J. Neurosurg. 82:269-277 (1995) subjectedrabbits to severe spinal cord ischemia (by inflating the balloon of acatheter tip which had been introduced in the abdominal aorta) followedby 30 minutes of reperfusion at which time either: (1) vehicle, (2)anti-CD18 antibody, or (3) anti-CD18 antibody and platelet-activatingfactor (PAF) antagonist were administered to the animals. Recovery ofmotor function was improved by the anti-CD18 antibody, but no furtherimprovement was induced by the PAF antagonist.

[0016] It has been observed that while an anti-CD18 antibody reducedneurologic deficits in the reversible spinal cord model (involving asnare ligature occluding device), it was unable to do so in anirreversible microsphere model. Clark et al., Stroke 22(7): 877-883(1991). Clark et al. conclude that leukocytes potentiate their effect incentral nervous system injury via reperfusion injury. With respect toanti-CD11b antibodies, Chopp et al., Stroke 25(1):267 (1994) report thatbenefit from administration of such antibodies was observed underconditions of transient, but not permanent, MCA occlusion in rats. See,also, Jiang et al., Neuroscience Research Communications 15(2):85-93(1994). Clark et al., J. Neurosurg 75(4):623-627 (1991) also observethat while anti-ICAM-1 produced a significant reduction in neurologicaldeficits in the reversible spinal cord ischemia model, such therapeuticbenefit was not seen in the irreversible brain ischemia model. Similarfindings in relation to anti-ICAM-1 antibodies have also been reportedby Zhang et al., Stroke 26(8):1438-1442 (1995).

[0017] Bowes et al., Neurology 45:815-819 (1995) evaluated the abilityof monoclonal antibodies directed against ICAM-1 and CD18 to enhance theefficacy of thrombolysis in a rabbit cerebral embolism stroke model. Inthis model, numerous small blood clots (formed by fragmenting a clotwith a tissue homogenizer) were injected into the rabbit's carotidcirculation in order to achieve embolization. Neurologic function ineach animal was evaluated 18 hours following embolization on a threepoint scale: (1) normal activity; (2) abnormal activity; or (3) death.The amount of clot necessary to produce permanent neurologic damage in50% of the rabbits (ED₅₀) was determined for each treatment group. Boweset al., found that when administration of anti-CD18 or anti-ICAM-1 wasdelayed until 15 or 30 minutes after embolization, a statisticallysignificant improvement in neurologic function was not observed. Seealso Bowes et al., Experimental Neurology 119(2):215-219 (1993) inrelation to earlier work by this group regarding anti-ICAM-1 and t-PA intheir rabbit cerebral embolism stroke model.

[0018] Bednar et al., Stroke 23(1):152 (1992) describe a rabbit model ofthromboembolic stroke wherein the arterial occlusion (an autologousblood clot delivered to the anterior cerebral circulation) is notremoved during the experiment. Rabbits received either anti-CD18antibody IB4 (1 mg/kg), or vehicle, 30 minutes following thethromboembolic event. Following embolization, the animals were studiedfor a total of 4 hours, including an initial 45 minutes of systemichypotension. No statistically significant difference in cerebral bloodflow (CBF) or infarct size between IB4 and vehicle treated animals wasseen. However, IB4 did attenuate intracranial hypertension in thismodel.

[0019] It is an object of the present invention to provide a method forimproving clinical outcome in acute ischemic stroke by increasingcerebral blood flow and/or reducing infarct size. Furthermore, it is anobject of the invention to provide an alternative to thrombolytictherapy for treating thromboembolic stroke, particularly wherethrombolytic therapy has been unsuccessful or is contra-indicated, as isthe case where the patient to be treated is taking aspirin, or where thetime delay from the onset of stroke to diagnosis is such thatthrombolytic therapy is not recommended. Other objects will be apparentfrom the description which follows.

SUMMARY OF THE INVENTION

[0020] This application is based on the unexpected finding thatanti-CD18 antibodies can lead to a significant increase in cerebralblood flow and/or reduction in brain infarct size in focal ischemicstroke, in the absence of removal of the arterial obstruction.

[0021] Accordingly, the invention provides a method for treating strokein a mammal (e.g. focal ischemic stroke caused by obstruction of a maincerebral artery) which comprises the step of administering an amount ofCD18 antagonist and/or CD11b antagonist to the mammal which is effectivefor increasing cerebral blood flow and/or reducing cerebral infarct sizein the mammal. In the method, the arterial obstruction (generally asingle thrombus or embolus) is not removed by mechanical means (e.g. bysurgically removing the obstruction) or chemical means (e.g. by using athrombolytic agent to dissolve the arterial obstruction). Furthermore,the recipient of the CD18 or CD11b antagonist is not subjected toextraneous systemic hypotension (e.g., via controlled exsanguination)during the method as was the case in Bednar et al., Stroke 23(1):152(1992).

[0022] Preferably the antagonist is an anti-CD18 antibody, such as ahumanized F(ab′)₂ fragment. Conveniently, the antagonist is administeredto the mammal in the form of a pharmaceutically acceptable formulation,such as those elaborated in more detail herein.

[0023] The preferred mode of administration of the antagonist is bybolus intravenous dosage. In certain embodiments of the invention, theantagonist may be administered at least once a time between about 15minutes to about 20 hours and preferably between about 30 minutes toabout 12 hours from the onset of focal ischemic stroke. Single ormultiple dosages may be given. Alternatively, or in addition, theantagonist may be administered via continuous infusion.

[0024] In another aspect, the invention relates to a method for treatingischemic stroke caused by systemic hypoperfusion or hypoxia in a mammal(e.g. resulting from cardiac arrest or drowning) which comprises thestep of administering a therapeutically effective amount of CD11bantagonist and/or CD18 antagonist to the mammal. The preferredantagonist for use in this method is an anti-CD18 antibody. Preferably,the method results in an increase in cerebral blood flow and a decreasein infarct size resulting from the systemic hypoperfusion.

[0025] In yet another embodiment, the invention provides a method forincreasing cerebral blood flow and/or reducing infarct size in focalischemic stroke caused by obstruction of a main cerebral artery whichcomprises the step of administering a therapeutically effective amountof CD18 and/or CD11b antagonist to the mammal at least once at a timemore than 15 mins (e.g. more than 30 mins) and preferably less than 24hours from the onset of focal ischemic stroke in the mammal. In themethod, the mammal being treated is not subjected to extraneous systemichypotension as described in Bednar et al., Stroke 23(1):152 (1992).Preferably the antagonist is an anti-CD18 antibody which is administeredat least once at a time between about 30 minutes to about 12 hours fromthe onset of focal ischemic stroke.

[0026] According to the method of the previous paragraph, atherapeutically effective amount of a thrombolytic agent (such as t-PA)may be co-administered to the mammal either before, after, orsimultaneously with, the CD11b or CD18 antagonist. Preferably, theantagonist is administered to the mammal prior to administration of thethrombolytic agent. According to this method, the thrombolytic agent maybe administered to the mammal more than about 3 hours after the onset ofischemic stroke (e.g., at least once within about 3-8 hours andpreferably within about 3-5 hours from the onset of stroke). Thethrombolytic agent may be administered by one or more bolus doses or bycontinuous infusion, for example.

[0027] The invention also provides articles of manufacture and kits foruse in the above methods.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIG. 1 is a bar graph depicting brain infarct size (% hemisphereinfarcted) in embolized rabbits following treatment with MHM23(anti-CD18) and t-PA (n=5); MHM23 alone (n=5); t-PA alone (n=10) orsaline solution control (n=10) as described in Example 1(mean+/−standard error of the mean).

[0029]FIG. 2 depicts regional cerebral blood flow (CBF; cc/100 gm/min)over time in embolized rabbits following treatment with MHM23(anti-CD18) and t-PA (n=5); MHM23 alone (n=5); t-PA alone (n=10) orsaline solution control (n=10) as described in Example 1. MHM23 orsaline solution control was administered 1 hour following embolization.t-PA or saline solution control was administered by continuous infusionover hours 3-5 following embolization (mean+/−standard error of themean).

[0030]FIG. 3 illustrates intracranial pressure (ICP; mm Hg) in embolizedrabbits following treatment with MHM23 (anti-CD18) and t-PA (n=5); MHM23alone (n=5); t-PA alone (n=10) or saline solution control (n=10) asdescribed in Example 1. MHM23 or saline solution control wasadministered 1 hour following embolization. t-PA or saline solutioncontrol was administered by continuous infusion over hours 3-5 followingembolization (mean+/−standard error of the mean).

[0031] FIGS. 4A-B depict an alignment of the relevant portions of theconsensus amino acid sequences of the human IgG₁ CH1 domain (SEQ ID NO:1), the human IgG₂CH1 domain (SEQ ID NO: 2), the human IgG₃ CH1 domain(SEQ ID NO: 3), the human IgG₄ CH1 domain (SEQ ID NO: 4), the human K CLdomain (SEQ ID NO: 5), and the human λ C_(L) domain (SEQ ID NO: 6), inalignment with the Fabvlb variant derived from an anti-CD18 antibody(SEQ ID NO: 7). In FIGS. 4A-B, amino acid residues and/or positions ofinterest and of most importance for use as salvage receptor bindingepitopes within the sequence of Fabvlb (i.e., SEQ ID NOS: 8 and 9) aredesignated by underlining and asterisks, respectively.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] I. Definitions

[0033] “Focal ischemic stroke” is defined herein as damage to the braincaused by interruption of the blood supply to a region thereof. Thefocal ischemic stroke of interest herein is generally caused byobstruction of any one or more of the “main cerebral arteries” (e.g.middle cerebral artery, anterior cerebral artery, posterior cerebralartery, internal carotid artery, vertebral artery or basilar artery), asopposed to secondary arteries or arterioles. The “arterial obstruction”is generally a single embolus or thrombus. Hence, focal ischemic strokeas defined herein is distinguished from the cerebral embolism strokemodel of Bowes et al., Neurology 45:815-819 (1995) in which a pluralityof clot particles occlude secondary arteries or arterioles.

[0034] The expression “in the absence of removal of the arterialobstruction” when used throughout this application means that thearterial obstruction is essentially not removed by mechanical means (forexample, by physically removing the obstruction as described in thetransient stroke models described above) or by chemical means (such asremoval of a thrombus orembolus using a thrombolytic agent; see, e.g.,Bowes et al., Neurology 45:815-819 (1995)) prior to a therapeuticbenefit achieved by administration of the CD18 or CD11b antagonist(i.e., the increase in cerebral blood flow and/or the reduction ininfarct size). However, this term would encompass situations wherein thearterial obstruction is slightly reduced in size as a consequence ofendogenous thrombolytic molecules dissolving part of thethrombus/embolus, provided that at least some (e.g., about 50% andpreferably about 80%) of the arterial obstruction remains intactfollowing any such size reduction.

[0035] By “increasing cerebral blood flow or reducing infarct size” ismeant the act of improving clinical outcome by inducing a statisticallyor physiologically significant increase in cerebral blood flow and/or astatistically or physiologically significant reduction in infarct sizein a treated mammal relative to an untreated mammal as determined usingtechniques which are well known in the art, such as vascular imaging,for example. Preferably cerebral blood flow as determined 2-4 hoursafter administration of the antagonist is increased by at least 30% andpreferably at least 50% relative to an untreated mammal. Desirably,infarct size measured 48 hours after administration of the antagonistwill be 20% less and preferably 50% less than that of an untreatedmammal.

[0036] The term “CD18 antagonist” when used herein refers to a moleculewhich binds to CD18 (preferably human CD18) and inhibits orsubstantially reduces a biological activity of CD18. Normally, theantagonist will block (partially or completely) the ability of a cell(e.g. a neutrophil) expressing the CD18 subunit at its cell surface tobind to endothelium. Non-limiting examples of CD18 antagonists includeantibodies, proteins, peptides, glycoproteins, glycopeptides,glycolipids, polysaccharides, oligosaccharides, nucleic acids,bioorganic molecules, peptidomimetics, pharmacological agents and theirmetabolites, transcriptional and translation control sequences, and thelike. In the preferred embodiment of the invention, the CD18 antagonistis an antibody.

[0037] Examples of anti-CD18 antibodies include MHM23 (Hildreth et al.,Eur. J. Immunol. 13:202-208 (1983)); M18/2 (IgG₂a; Sanches-Madrid et al,J. Exp. Med. 158:586 (1983)); H52 (American Type Culture Collection(ATCC) Deposit HB 10160); Mas191c and IOT18 (Vermot Desroches et al.,Scand. J. Immunol. 33:277-286 (1991)); and NA-8 (WO 94/12214). Thepreferred antibody is one which binds to the CD18 epitope to whicheither MHM23 or H52 binds. Preferably the antibody has a high affinityfor the CD18 polypeptide. In preferred embodiments, the antibody has anaffinity for the CD18 antigen of about 4 nM or less. Preferably, theaffinity is about 3 nM or less, and most preferably 1 nM or less. Incertain embodiments, the antibody may bind to a region in theextracellular domain of CD18 which associates with CD11b and theantibody may also dissociate α and β chains (e.g. the antibody maydissociate the CD11b and CD18 complex as is the case for the MHM23antibody).

[0038] The term “CD11b antagonist” when used herein refers to a moleculewhich binds to CD11b and inhibits or substantially reduces a biologicalactivity of CD11b. Normally, the antagonist will block (partially orcompletely) the ability of a cell (e.g. a neutrophil) expressing theCD11b subunit at its cell surface to bind to endothelium. Non-limitingexamples of CD11b antagonists include antibodies, proteins, peptides,glycoproteins, glycopeptides, glycolipids, polysaccharides,oligosaccharides, nucleic acids, bioorganic molecules, peptidomimetics,pharmacological agents and their metabolites, transcriptional andtranslation control sequences, and the like. The preferred CD11bantagonist is an antibody, especially an anti-CD11b antibody which bindshuman CD11b. Exemplary CD11b antibodies include MY904 (U.S. Pat. No.4,840,793); 1B6c (see Zhang et al., Brain Research 698:79-85 (1995));CBRN1/5 and CBRM1/19 (WO94/08620).

[0039] The term “antibody” is used in the broadest sense andspecifically covers monoclonal antibodies, antibody compositions withpolyepitopic specificity, bispecific antibodies, diabodies, andsingle-chain molecules, as well as antibody fragments (e.g., Fab,F(ab′)₂, and Fv), so long as they antagonize the biological activity ofCD11b or CD18.

[0040] The term “monoclonal antibody” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site. Furthermore, in contrastto conventional (polyclonal) antibody preparations which typicallyinclude different antibodies directed against different determinants(epitopes), each monoclonal antibody is directed against a singledeterminant on the antigen. In addition to their specificity, themonoclonal antibodies are advantageous in that they are synthesized bythe hybridoma culture, uncontaminated by other immunoglobulins. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler et al., Nature, 256: 495 (1975), or maybe made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

[0041] The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;Morrison et al., Proc. Nat. Acad. Sci. USA, 81:6851-6855 (1984)).

[0042] “Humanized” forms of non-human (e.g., murine) antibodies arechimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementary-determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity, andcapacity. In some instances, Fv framework region (FR) residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. These modifications are made to further refine and optimizeantibody performance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin sequence. The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature,321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol., 2:593-596 (1992). The humanizedantibody includes a Primatized™ antibody wherein the antigen-bindingregion of the antibody is derived from an antibody produced byimmunizing macaque monkeys with the antigen of interest.

[0043] “Single-chain Fv” or “sFv” antibody fragments comprise the V_(H)and V_(L) domains of antibody, wherein these domains are present in asingle polypeptide chain. Generally, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding. For areview of sFv see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

[0044] The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

[0045] As used herein, the term “salvage receptor binding epitope”refers to an epitope of the Fc region of an IgG molecule (e.g., IgG₁,IgG₂, IgG₃, and IgG₄) that is responsible for increasing the in vivoserum half-life of the IgG molecule.

[0046] “Treatment” refers to both therapeutic treatment and prophylacticor preventative measures. Those in need of treatment include thosealready with the disorder as well as those in which the disorder is tobe prevented. This application is mostly concerned with treating thoseindividuals who have been diagnosed as having suffered acute ischemicstroke.

[0047] “Mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.Preferably, the mammal is human.

[0048] A “thrombolytic agent” is a molecule which breaks up or dissolvesa thrombus. Exemplary thrombolytic agents include streptokinase,acylated plasminogen-streptokinase activator complex (APSAC), urokinase,single-chain urokinase-plasminogen activator (scuPA), thrombinlikeenzymes from snake venoms such as ancrod (Bell, W. “Defribinogenatingenzymes” In Colman et al., (eds): Hemostasis and Thrombosis Lippincott,Philadelphia (1987) p.886), tissue plasminogen activator (t-PA) andbiologically active variants of each of the above. The preferredthrombolytic agent is t-PA.

[0049] In the context of the present invention, the terms “tissueplasminogen activator” and “t-PA” are used interchangeably and denoteextrinsic (tissue type) plasminogen activator having at least twofunctional domains consisting of a protease domain that is capable ofconverting plasminogen to plasmin and an N-terminal region believed tobe responsible for fibrin binding. These terms therefore includepolypeptides containing these functional domains as part of the overallamino acid sequence, irrespective of their source and method ofpreparation (e.g. these terms cover vampire bat t-PAs as disclosed in EP352,119). The terms “human tissue plasminogen activator” and “humant-PA” are used interchangeably and denote wild-type human tissueplasminogen activator and functional derivatives thereof. Examples oft-PA functional derivatives include those molecules with extendedhalf-life and improved fibrin specificity as disclosed in WO 93/24635;N-terminally truncated t-PA variants (see EP 382,174); and C84S t-PAdescribed in Suzuki et al. J. Cardiovasc. Pharmacol. 22:834-840 (1993),for example.

[0050] II. Modes for Carrying Out the Invention

[0051] The invention provides a method for treating focal ischemicstroke, such as thromoboembolic stroke. In particular, cerebral bloodflow can be increased and/or infarct size can be reduced in focalischemic stroke by administering an effective amount of a CD11b and/orCD18 antagonist to the mammal having suffered the stroke. In thismethod, the arterial obstruction is not removed prior to observation ofthe therapeutic benefit as defined herein, and as such the method doesnot require prior administration of a thrombolytic agent to the mammalin order to remove an embolus/thrombus and thereby increase cerebralblood flow and/or reduce infarct size.

[0052] It is contemplated that the CD18 or CD11a antagonist of thepresent invention will be administered to a patient as soon as possibleonce the condition of acute ischemic stroke has been diagnosed or issuggested by focal deficit on neurologic examination. Neurologicexamination and, optionally, neuroimaging techniques such as computedtomography (CT) and magnetic resonance imaging (MRI) (includingdiffusion weighted imaging (DWI) and perfusion imaging (PI)); vascularimaging (e.g., duplex scanning and transcranial Doppler ultrasound andlaser Doppler); angiography (e.g., computerized digital subtractionangiography (DSA) and MR angiography) as well as other invasive ornon-invasive techniques are available for the diagnosis of acuteischemic stroke.

[0053] Preferably, the CD18 or CD11a antagonist will be administered atleast once or continuously at any time from immediately following toabout 24 hours after the onset of stroke. In certain embodiments, theCD18 or CD11a antagonist is first administered to the patient at a timebetween about 15 minutes (or 30 minutes or 45 minutes) to about 5 hours(or 12 hours or 24 hours) from the onset of stroke. For example, theantagonist may be first administered by bolus dosage as soon as strokeis diagnosed, followed by a subsequent bolus dosage of the antagonist(e.g. 5-24 hours after the initial bolus dosage).

[0054] The preferred antagonist for use in the above method is humanizedH52 antibody (huH52), especially the huH52 F(ab′)₂ antibody fragment.

[0055] The sequence of the heavy chain of the huH52 Fab is:EVQLVESGGGLVQPGGSLRLSCATSGYTFTEYTMHWMRQAPGKGLEWVAGINPKNG (SEQ ID NO:10)GTSHNQRFMDRFTISVDKSTSTAYMQMNSLRAEDTAVYYCARWRGLNYGFDVRYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THT.

[0056] The sequence of the light chain of the huH52 Fab is:DIQMTQSPSSLSASVGDRVTITCRASQDINNYLNWYQQKPGKAPKLLIYYTSTLHSGVP (SEQ IDNO:11) SRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0057] In other embodiments, the full length IgG₂ huH52 antibody may bethe molecule of choice. The heavy chain of the full length IgG₂ huH52antibody has the sequence:EVQLVESGGGLVQPGGSLRLSCATSGYTFTEYTMHWMRQAPGKGLEWVAGINPKNG (SEQ ID NO:12)GTSHNQRFMDRFTISVDKSTSTAYMQMNSLRAEDTAVYYCARWRGLNYGFDVRYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTWHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0058] The light chain of the full length IgG₂ huH52 antibody has thesequence: DIQMTQSPSSLSASVGDRVTITCRASQDINNYLNWYQQKPGKAPKLLIYYTSTLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC.

[0059] A description follows as to the production of the preferredantagonists (i.e. antibodies), long half-life antagonists,pharmaceutical formulations, modes for administration, as well as kitsand articles of manufacture for use in the claimed methods. Thedescription in relation to long half-life antagonists, pharmaceuticalformulations and modes for administration is also relevant to the use ofthrombolytic agents in certain embodiments of the invention.

[0060] A. Antibody Preparation

[0061] According to the preferred embodiment of the invention, the CD18or CD11b antagonist is an antibody. Various antibodies which bind toCD18 and CD11b are available in the art. Furthermore, a descriptionfollows as to the production of anti-CD18 or anti-CD11b antibodies foruse in the treatment of stroke as defined herein.

[0062] (i) Polyclonal Antibodies.

[0063] Polyclonal antibodies are generally raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), Nhydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R′ are different alkyl groups.

[0064] Animals are immunized against the antigen, immunogenicconjugates, or derivatives by combining 1 mg or 1 μg of the peptide orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with ⅕ to {fraction(1/10)} the original amount of peptide or conjugate in Freund's completeadjuvant by subcutaneous injection at multiple sites. Seven to 14 dayslater the animals are bled and the serum is assayed for antibody titer.Animals are boosted until the titer plateaus. Preferably, the animal isboosted with the conjugate of the same antigen, but conjugated to adifferent protein and/or through a different cross-linking reagent.Conjugates also can be made in recombinant cell culture as proteinfusions. Also, aggregating agents such as alum are suitably used toenhance the immune response.

[0065] (ii) Monoclonal Antibodies.

[0066] Monoclonal antibodies are obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts. Thus, themodifier “monoclonal” indicates the character of the antibody as notbeing a mixture of discrete antibodies.

[0067] For example, the monoclonal antibodies may be made using thehybridoma method first described by Kohler et al., Nature, 256:495(1975), or may be made by recombinant DNA methods (U.S. Pat. No.4,816,567).

[0068] In the hybridoma method, a mouse or other appropriate hostanimal, such as a hamster, is immunized as hereinabove described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)).

[0069] The hybridoma cells thus prepared are seeded and grown in asuitable culture medium that preferably contains one or more substancesthat inhibit the growth or survival of the unfused, parental myelomacells. For example, if the parental myeloma cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (HAT medium), which substances prevent thegrowth of HGPRT-deficient cells.

[0070] Preferred myeloma cells are those that fuse efficiently, supportstable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these, preferred myeloma cell lines are murine myelomalines, such as those derived from MOPC-21 and MPC-11 mouse tumorsavailable from the Salk Institute Cell Distribution Center, San Diego,Calif. USA, and SP-2 cells available from the American Type CultureCollection, Rockville, Md. USA. Human myeloma and mouse-humanheteromyeloma cell lines also have been described for the production ofhuman monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984);Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0071] Culture medium in which hybridoma cells are growing is assayedfor production of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

[0072] The binding affinity of the monoclonal antibody can, for example,be determined by the Scatchard analysis of Munson et al., Anal.Biochem., 107:220 (1980).

[0073] After hybridoma cells are identified that produce antibodies ofthe desired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

[0074] The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

[0075] DNA encoding the monoclonal antibodies is readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells serveas a preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells suchas E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells,or myeloma cells that do not otherwise produce immunoglobulin protein,to obtain the synthesis of monoclonal antibodies in the recombinant hostcells. Review articles on recombinant expression in bacteria of DNAencoding the antibody include Skerra et al., Curr. Opinion in Immunol.,5:256-262 (1993) and Pluckthun, Immunol. Revs., 130:151-188 (1992).

[0076] In a further embodiment, antibodies or antibody fragments can beisolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554(1990). Clackson etal., Nature, 352:624-628(1991) and Marks et al., J. Mol. Biol.,222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse et al., Nuc.Acids. Res., 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

[0077] The DNA also may be modified, for example, by substituting thecoding sequence for human heavy- and light-chain constant domains inplace of the homologous murine sequences (U.S. Pat. No. 4,816,567(Morrison, et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide.

[0078] Typically such non-immunoglobulin polypeptides are substitutedfor the constant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

[0079] Chimeric or hybrid antibodies also may be prepared in vitro usingknown methods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide-exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

[0080] (iii) Humanized and Human Antibodies.

[0081] Methods for humanizing non-human antibodies are well known in theart. Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567, wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

[0082] The choice of human variable domains, both light and heavy, to beused in making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987)). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).

[0083] It is further important that antibodies be humanized withretention of high affinity for the antigen and other favorablebiological properties. To achieve this goal, according to a preferredmethod, humanized antibodies are prepared by a process of analysis ofthe parental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

[0084] Alternatively, it is now possible to produce transgenic animals(e.g., mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits etal., Proc. Natl. Acad. Sci. USA, 90:2551(1993);Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Yearin Immuno., 7:33 (1993). Human antibodies can also be derived fromphage-display libraries (Hoogenboom et al., J. Mol. Biol., 227:381(1991); Marks et al., J. Mol. Biol., 222:581-597 (1991)).

[0085] (iv) Bispecific Antibodies

[0086] Bispecific antibodies (BsAbs) are antibodies that have bindingspecificities for at least two different epitopes. Exemplary BsAbs maybind to two different epitopes of the CD18 antigen or may bind both CD18and CD11b. Such antibodies can be derived from full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies).

[0087] Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, published May 13, 1993, and inTraunecker et al., EMBO J., 10:3655-3659 (1991).

[0088] According to a different and more preferred approach, antibodyvariable domains with the desired binding specificities(antibody-antigen combining sites) are fused to immunoglobulin constantdomain sequences. The fusion preferably is with an immunoglobulin heavychain constant domain, comprising at least part of the hinge, CH2, andCH3 regions. It is preferred to have the first heavy-chain constantregion (CH1) containing the site necessary for light chain binding,present in at least one of the fusions. DNAs encoding the immunoglobulinheavy chain fusions and, if desired, the immunoglobulin light chain, areinserted into separate expression vectors, and are co-transfected into asuitable host organism. This provides for great flexibility in adjustingthe mutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

[0089] In a preferred embodiment of this approach, the bispecificantibodies are composed of a hybrid immunoglobulin heavy chain with afirst binding specificity in one arm, and a hybrid immunoglobulin heavychain-light chain pair (providing a second binding specificity) in theother arm. It was found that this asymmetric structure facilitates theseparation of the desired bispecific compound from unwantedimmunoglobulin chain combinations, as the presence of an immunoglobulinlight chain in only one half of the bispecific molecule provides for afacile way of separation. This approach is disclosed in WO 94/04690published Mar. 3, 1994. For further details of generating bispecificantibodies see, for example, Suresh et al., Methods in Enzymology,121:210 (1986). Using such techniques, a bispecific molecule whichcombines a thrombolytic agent such as t-PA and an anti-CD18 oranti-CD11b antibody can be prepared for use in the treatment of strokeas defined herein.

[0090] Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

[0091] Techniques for generating bispecific antibodies from antibodyfragments have also been described in the literature. The followingtechniques can also be used for the production of bivalent antibodyfragments which are not necessarily bispecific. For example, Fab′fragments recovered from E. coli can be chemically coupled in vitro toform bivalent antibodies. See, Shalaby et al., J. Exp. Med., 175:217-225(1992).

[0092] Various techniques for making and isolating bivalent antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bivalent heterodimers have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. The “diabody”technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA,90:6444-6448 (1993) has provided an alternative mechanism for makingbispecific/bivalent antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific/bivalentantibody fragments by the use of single-chain Fv (sFv) dimers has alsobeen reported. See Gruber et al., J. Immunol., 152:5368 (1994).

[0093] B. Long Half-Life Antagonists

[0094] In certain embodiments of the invention, it is desirable to useCD18 or CD11b antagonists engineered to have an enhanced half-life inthe serum of a mammal treated therewith. For example, this may beachieved by (i) incorporating a salvage receptor binding epitope of theFc region of an IgG into the antagonist so as to increase itscirculatory half-life, but without disrupting its biological activity or(ii) covalently binding a nonproteinaceous polymer to the antagonist.These exemplary techniques will be described briefly below:

[0095] (i) Antagonist-Salvage Receptor Binding Epitope Fusions.

[0096] Incorporation of a salvage receptor binding epitope into theantagonist can take place by any means, such as by mutation of theappropriate region in the antagonist of interest to mimic the Fc regionor by incorporating the epitope into a peptide tag that is then fused tothe antagonist at either end or in the middle or by DNA or peptidesynthesis.

[0097] A systematic method for preparing such an antagonist varianthaving an increased in vivo half-life comprises several steps. The firstinvolves identifying the sequence and conformation of a salvage receptorbinding epitope of an Fc region of an IgG molecule. Once this epitope isidentified, the sequence of the antagonist of interest is modified toinclude the sequence and conformation of the identified binding epitope.After the sequence is mutated, the antagonist variant is tested to seeif it has a longer in vivo half-life than that of the originalantagonist. If the antagonist variant does not have a longer in vivohalf-life upon testing, its sequence is further altered to include thesequence and conformation of the identified binding epitope. The alteredantagonist is tested for longer in vivo half-life, and this process iscontinued until a molecule is obtained that exhibits a longer in vivohalf-life.

[0098] The salvage receptor binding epitope being thus incorporated intothe antagonist of interest is any suitable such epitope as definedabove, and its nature will depend, e.g., on the type of antagonist beingmodified. The transfer is made such that the antagonist of interest isstill able to antagonize the biological activity of CD11b or CD18.

[0099] Where the antagonist of interest is an antibody, it contains anIg domain or Ig-like domain and the salvage receptor binding epitope isplaced so that it is located within this Ig domain or Ig-like domain.More preferably, the epitope constitutes a region wherein any one ormore amino acid residues from one or two loops of the Fc domain aretransferred to an analogous position of the Ig domain or Ig-like domainof the antibody. Even more preferably, three or more residues from oneor two loops of the Fc domain are transferred. Still more preferred, theepitope is taken from the CH2 domain of the Fc region (e.g., of an IgG)and transferred to the CH1, CH3, or VH region, or more than one suchregion, of an Ig or to a Ig-like domain. Alternatively, the epitope istaken from the CH2 domain of the Fc region and transferred to the CLregion or VL region, or both, of an Ig or to an Ig-like domain of theantagonist of interest.

[0100] For example, for purposes of discussing variants wherein thepolypeptide of interest is an antibody, reference is made to FIGS. 4A-B,which illustrates the relevant consensus primary structures of variousIgs, i.e., human IgG₁ CH1 domain, human IgG₂ CH1 domain, human IgG₃ CH1domain, human IgG₄ CH1 domain, human κC_(L) domain, and human λC_(L)domain, as well as the specific sequence for Fabv1b, a preferredanti-CD18 Fab variant herein. Further, FIGS. 4A-B indicates the residuesof Fabv1b that are of interest and of most importance. In a preferredembodiment, the residues of importance are those with an asterisk inFIGS. 4A-B, ie., in one loop of Fabv1b, MIS with a T residue one aminoacid C-terminal to MIS, and in another loop of Fabv1b, HQN with a Dresidue two amino acids C-terminal to HQN and a K residue one amino acidC-terminal to the D residue.

[0101] In one most preferred embodiment, the salvage receptor bindingepitope comprises the sequence (5′ to 3′): PKNSSMISNTP (SEQ ID NO:8),and optionally further comprises a sequence selected from the groupconsisting of HQSLGTQ (SEQ ID NO: 13), HQNLSDGK (SEQ ID NO:9), HQNISDGK(SEQ ID NO:14), or VISSHLGQ (SEQ ID NO:15), particularly where theantagonist of interest is a Fab or F(ab′)₂.

[0102] (ii) Antagonist-Polymer Conjugates.

[0103] The nonproteinaceous polymer of choice for this purpose isordinarily is a hydrophilic synthetic polymer, i.e., a polymer nototherwise found in nature. However, polymers which exist in nature andare produced by recombinant or in vitro methods are useful, as arepolymers which are isolated from native sources. Hydrophilic polyvinylpolymers fall within the scope of this invention, e.g. polyvinylalcoholand polyvinylpyrrolidone. Particularly useful are polyalkylene etherssuch as polyethylene glycol (PEG); polyelkylenes such aspolyoxyethylene, polyoxypropylene, and block copolymers ofpolyoxyethylene and polyoxypropylene (Pluronics); polymethacrylates;carbomers; branched or unbranched polysaccharides which comprise thesaccharide monomers D-mannose, D- and L-galactose, fucose, fructose,D-xylose, L-arabinose, D-glucuronic acid, sialic acid, D-galacturonicacid, D-mannuronic acid (e.g. polymannuronic acid, or alginic acid),D-glucosamine, D-galactosamine, D-glucose and neuraminic acid includinghomopolysaccharides and heteropolysaccharides such as lactose,amylopectin, starch, hydroxyethyl starch, amylose, dextrane sulfate,dextran, dextrins, glycogen, or the polysaccharide subunit of acidmucopolysaccharides, e.g. hyaluronic acid; polymers of sugar alcoholssuch as polysorbitol and polymannitol; heparin or heparon. The polymerprior to cross-linking need not be, but preferably is, water soluble,but the final conjugate must be water soluble. In addition, the polymershould not be highly immunogenic in the conjugate form, nor should itpossess viscosity that is incompatible with intravenous infusion orinjection if it is intended to be administered by such routes.

[0104] Preferably the polymer contains only a single group which isreactive. This helps to avoid cross-linking of protein molecules.However, it is within the scope herein to optimize reaction conditionsto reduce cross-linking, or to purify the reaction products through gelfiltration or chromatographic sieves to recover substantially homogenousderivatives.

[0105] The molecular weight of the polymer may desirably range fromabout 100 to 500,000, and preferably is from about 1,000 to 20,000. Themolecular weight chosen will depend upon the nature of the polymer andthe degree of substitution. In general, the greater the hydrophilicityof the polymer and the greater the degree of substitution, the lower themolecular weight that can be employed. Optimal molecular weights will bedetermined by routine experimentation.

[0106] The polymer generally is covalently linked to the antagonistthough a multifunctional crosslinking agent which reacts with thepolymer and one or more amino acid or sugar residues of the antagonistto be linked. However, it is within the scope of the invention todirectly crosslink the polymer by reacting a derivatized polymer withthe hybrid, or vice versa.

[0107] Covalent binding to amino groups is accomplished by knownchemistries based upon cyanuric chloride, carbonyl diimidazole, aldehydereactive groups (PEG alkoxide plus diethyl acetal of bromoacetaldehyde;PEG plus DMSO and acetic anhydride, or PEG chloride plus the phenoxideof 4-hydroxybenzaldehyde, succinimidyl active esters, activateddithiocarbonate PEG, 2,4,5-trichlorophenylcloroformate orP-nitrophenylcloroformate activated PEG). Carboxyl groups arederivatized by coupling PEG-amine using carbodiimide.

[0108] Polymers are conjugated to oligosaccharide groups by oxidationusing chemicals, e.g. metaperiodate, or enzymes, e.g. glucose orgalactose oxidase, (either of which produces the aldehyde derivative ofthe carbohydrate), followed by reaction with hydrazide or aminoderivatized polymers, in the same fashion as is described by Heitzmannet al., Proc. Natl. Acad. Sci. USA 71:3537-41 (1974) or Bayer et al.,Methods in Enzymology 62:310 (1979), for the labeling ofoligosaccharides with biotin or avidin. Further, other chemical orenzymatic methods which have been used heretofore to linkoligosaccharides are particularly advantageous because, in general,there are fewer substitutions than amino acid sites for derivatization,and the oligosaccharide products thus will be more homogenous. Theoligosaccharide substituents also are optionally modified by enzymedigestion to remove sugars, e.g. by neuraminidase digestion, prior topolymer derivatization.

[0109] The polymer will bear a group which is directly reactive with anamino acid side chain, or the N- or C-terminus of the antagonist linked,or which is reactive with the multifunctional cross-linking agent. Ingeneral, polymers bearing such reactive groups are known for thepreparation of immobilized proteins. In order to use such chemistrieshere, one should employ a water soluble polymer otherwise derivatized inthe same fashion as insoluble polymers heretofore employed for proteinimmobilization. Cyanogen bromide activation is a particularly usefulprocedure to employ in crosslinking polysaccharides.

[0110] “Water soluble” in reference to the starting polymer means thatthe polymer or its reactive intermediate used for conjugation issufficiently water soluble to participate in a derivatization reaction.“Water soluble” in reference to the polymer conjugate means that theconjugate is soluble in physiological fluids such as blood.

[0111] The degree of substitution with such a polymerwill vary dependingupon the number of reactive sites on the antagonist, whether all or afragment of the antagonist is used, whether the antagonist is a fusionwith a heterologous protein (e.g. anti-CD18 antibody fused to a salvagereceptor binding epitope), the molecular weight, hydrophilicity andother characteristics of the polymer, and the particular antagonistderivatization sites chosen. In general, the conjugate contains aboutfrom 1 to 10 polymer molecules, while any heterologous sequence may besubstituted with an essentially unlimited number of polymer molecules solong as the desired activity is not significantly adversely affected.The optimal degree of cross-linking is easily determined by anexperimental matrix in which the time, temperature and other reactionconditions are varied to change the degree of substitution, after whichthe ability of the conjugates to function in the desired fashion isdetermined.

[0112] The polymer, e.g. PEG, is cross-linked by a wide variety ofmethods known per se for the covalent modification of proteins withnonproteinaceous polymers such as PEG.

[0113] The long half-life conjugates of this invention are separatedfrom the unreacted starting materials by gel filtration. Heterologousspecies of the conjugates are purified from one another in the samefashion. The polymer also may be water-insoluble, as a hydrophilic gel.

[0114] C. Pharmaceutical Formulations

[0115] Therapeutic formulations of the CD11b or CD18 antagonist areprepared for storage by mixing the antagonist having the desired degreeof purity with optional physiologically acceptable carriers, excipientsor stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol,A. Ed. (1980)), in the form of lyophilized formulations or aqueoussolutions. Acceptable carriers, excipients or stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate and other organic acids; antioxidantsincluding ascorbic acid; low molecular weight (less than about 10residues) polypeptides; proteins, such as serum albumin, gelatin orimmunoglobulins; amino acids such as glycine, glutamine, asparagine,histidine, arginine or lysine; monosaccharides, disaccharides and othercarbohydrates including glucose, mannose, trehalose or dextrins;chelating agents such as EDTA; sugar alcohols such as mannitol orsorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as Tween, Pluronics or PEG.

[0116] The active ingredients may also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0117] The formulations to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes, prior to or following lyophilization andreconstitution.

[0118] Sustained-release preparations may be prepared. Suitable examplesof sustained-release preparations include semipermeable matrices ofsolid hydrophobic polymers containing the CD18 or CD11b antagonist,which matrices are in the form of shaped articles, e.g. films, ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate),or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919),copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acidglycolic acid copolymerssuch as the Lupron Depot™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated antibodies remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thiodisulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

[0119] Sustained-release CD18 or CD11b antagonist compositions alsoinclude liposomally entrapped antagonists. Liposomes containing the CD18or CD11b antagonist are prepared by methods known in the art, such asdescribed in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985);Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030 (1980); and U.S. Pat.Nos. 4,485,045 and 4,544,545. Ordinarily, the liposomes are the small(about 200-800 Angstroms) unilamelar type in which the lipid content isgreater than about 30 mol. % cholesterol, the selected proportion beingadjusted for the optimal CD18 or CD11b antagonist therapy. Liposomeswith enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.

[0120] D. Modes for Administration

[0121] The CD18 or CD11b antagonists of the invention are administeredto a mammal, preferably a human, in accord with known methods, such asintravenous administration as a bolus or by continuous infusion over aperiod of time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. Intravenous administration of theantagonist is preferred.

[0122] The appropriate dosage of CD18 or CD11b antagonist will depend onthe nature of the stroke to be treated, the severity and course of thestroke, whether the antagonist is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the CD18 or CD11b antagonist, and the discretion of theattending physician. The CD18 or CD11b antagonist is suitablyadministered to the patient at one time or over a series of treatmentsand may be administered to the patient at any time from diagnosisonwards. For example, the antagonist may be administered at a timebetween about 15, 30 or 45 minutes to about 5 hours, 12 hours or 24hours from the onset of stroke. In preferred embodiments, the initialdose is followed by at least one subsequent dose (e.g., from 5 to 24hours after the initial dose). In certain situations, CD11b antagonistand CD18 antagonist are co-administered to the mammal.

[0123] Where the antagonist is an antibody, from about 1100 μg/kg toabout 20 mg/kg, and preferably from about 500 μg/kg to about 5 mg/kg,and most preferably from about 1 mg/kg to about 3 mg/kg of the anti-CD18or anti-CD11b antibody is an initial candidate dosage for administrationto the patient, whether, for example, by one or more separateadministrations, or by continuous infusion. However, other dosageregimens may be useful. The progress of this therapy is easily monitoredby conventional techniques and assays elaborated herein.

[0124] E. Kits and Articles of Manufacture

[0125] In another embodiment of the invention, there are providedarticles of manufacture and kits containing materials useful forimproving clinical outcome in stroke by increasing cerebral blood flowand/or reducing infarct size. The article of manufacture comprises acontainer with a label. Suitable containers include, for example,bottles, vials, and test tubes. The containers may be formed from avariety of materials such as glass or plastic. The container holds acomposition which is effective for treating stroke as defined herein andmay have a sterile access port (for example the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). The active agent in the composition is aCD11b or CD18 antagonist. The label on the container indicates that thecomposition is used for treating stroke as described above, and may alsoindicate directions for in vivo use, such as those described above.

[0126] The kit of the invention comprises the container described aboveand a second container comprising a pharmaceutically-acceptable buffer,such as phosphate-buffered saline, Ringer's solution and dextrosesolution. It may further include other materials desirable from acommercial and user standpoint, including other buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse.

[0127] The following examples are offered by way of illustration and notby way of limitation. The disclosures of all citations in thespecification are expressly incorporated herein by reference.

EXAMPLE

[0128] This study investigated the effect of anti-CD18 antibody (MHM23)and t-PA in a rabbit model of thromboembolic stroke. In this model, asingle blood clot is introduced into the middle cerebral and posteriorcommunicating arteries (which are “main cerebral arteries”). Thearterial obstruction (i.e., the clot) remains in place throughout theexperiment (unless it is enzymatically removed by t-PA). The followingrabbit model is thought to correlate well with the physiologicalprogression of thromboembolic stroke in humans.

Materials and Methods

[0129] The rabbit model of thromboembolic stroke used in the currentstudy has been previously described in detail (Bednar et al., Neurol.Res., 16:129-132 (1994); Gross etal., Stroke, 24:558-562 (1993); Kohutet al., Stroke, 23:93-97 (1992); Wilson et al., Neurosurgery, 31:929-934(1992)). See, also, Gross et al., Neurosurgery, 36(6): 1172-1177 (1995).

[0130] Briefly, New Zealand white rabbits (Charles River, Calif.) (bothmales and females; 3.0-3.5 kg) were anesthetized with the solution ofketamine (50 mg/kg; Aveco Co., Fort Dodge, Iowa), acepromazine (20 mg,Aveco Co.), and xylazine (5 mg/kg; Mobay Corp., Shawnee, Kans.), asolution that was subsequently used to maintain sufficient anesthesiafor painless surgery (as determined by responses to variousphysiological and autonomic stimuli, including mean arterial pressureand response to a paw being pinched). After an incision was made in theright femoral triangle to expose the femoral vein and artery, thefemoral artery was cannulated with a PE-90 catheter (BD Co., Parsippany,N.J.), to which was attached a platinum-iridium electrode. Thiscatheter-electrode permitted the continuous measuring of mean arterialpressure and blood sampling for measurement of arterial blood gases(ABG) (pH, PCO₂, PO₂), hematocrit, and glucose and for determination ofhydrogen washout to assess the rCBF by the hydrogen clearance technique(Young, Stroke, 11:552-564 (1980)). After the femoral vein wascannulated with PE-90 tubing for drug infusions, a midline scalpincision was made to expose the calvarium. Bilateral craniectomies wereperformed and the following were placed; 30-gauge platinum-iridiumelectrodes to monitor the regional cerebral blood flow (rCBF); afiberoptic, epidural intracranial pressure (ICP) monitor (PrincetonMedical Corp., Hudson, N.H.); and a temperature sensor (Yellow SpringsInstruments, Yellow Springs, Ohio) to measure brain temperature. Allcranial instrumentation was carefully fixed in place with fast-settingepoxy. Through a midline neck incision, the animal was tracheostomizedand mechanically ventilated. Both depth and rate of ventilation weremodified as needed to maintain ABGs within physiological range.

[0131] Throughout the experiment, the brain and core temperatures, meanarterial pressure, and ICP were continuously measured. Additionally, thefollowing parameters were measured before embolization (baseline), atthe time of embolization, and hourly after embolization; the rCBF,hematocrit, glucose, and ABG. The mean arterial pressures were keptbetween 50 and 60 mm Hg throughout the experiment. Fluids (Ringer'slactate or packed cells) were given intravenously as needed(approximately 2-4 ml/kg/h) to maintain euvolemia. Core and braintemperatures were maintained within 1 C of baseline by using heatingblankets and heating lamps.

[0132] The autologous clot was prepared by mixing the whole blood (1 ml)with 50 mg of tin granules. The clot was introduced into the PE-90tubing pretreated with thrombin and was allowed to mature at roomtemperature.

[0133] After tracheostomy, the region of the bifurcation of the commoncarotid artery was identified, followed by 30 to 60 minutes ofequilibration, during which baseline values were obtained. All surgerywas completed within 2 hours. Once all the baseline values wereobtained, the proximal internal carotid artery and the distal commoncarotid artery were transiently isolated from the circulation. Anarteriotomy was then performed, and the autologous clot embolus wasdelivered to the anterior circulation of the brain via a catheteradvanced into the proximal internal carotid artery. Once embolized, boththe proximal internal carotid artery and distal common carotid arterywere again isolated from the circulation and an arteriorrhaphy wasperformed by using 10-0 interrupted nylon sutures. A Philip's dentalx-ray machine was used to obtain a submental-vertex radiograph thatverified placement of the tin-tagged clot. Embolized clots were notedwithin the middle cerebral and posterior communicating arteries.

[0134] t-PA ora saline solution (0.9% saline) was administeredintravenously by continuous infusion from hours 3-5 after theembolization at a total dose of 6.3 mg/kg. MHM23 (2 mg/kg) wasadministered by bolus dosage 1 hour after embolization. In eachinstance, the experiment continued for 7 hours after embolization.Submental-vertex radiographs were obtained after embolization and at theend of the experiment. Immediately after the embolization, the rCBF wasmeasured again; the experiment was continued if the rCBF was <15 ml/100g/min in any of the three electrodes in the embolized hemisphere (Joneset al., J. Neurosurg., 54:773-782 (1981)).

[0135] At the end of the experiment, the animals were killed with anoverdose of sodium pentobarbital (150 mg/kg), a procedure recognized asacceptable and painless, according to the euthanasia guidelines of theAmerican Veterinary Medical Association. Bilateral thoracotomies wereperformed in accordance with procedures outlined by the University ofVermont Institutional Animal Care and Utilization Committee. The brainwas harvested rapidly and examined grossly for the presence and positionof residual clot. The brain was cut into 2-mm slices in a bread-loaffashion and incubated in triphenyltetrazolium chloride dye to define thesize of the brain infarct (Bose et al., Stroke, 19:28-37 (1988)). Thismethod has been shown to be an acceptable means of determining the sizeof a brain infarct in our rabbit model and correlates well withhematoxylin and eosin staining (Bednar et al., Neurol. Res., 16:129-132(1994)). Each brain slice was carefully traced onto clear acetate sheetsfor later planimetric determination of the infarct size, for which anIBM image analyzer was used. The infarct size was determined accordingto the modification described by Lin et al., Stroke, 24:117-121 (1993).In this method, the region of the infarct is determined by subtractingthe volume of the noninfarcted part of the embolized hemisphere from theentire volume of the nonembolized hemisphere. This modification takesinto account that the volume of a brain infarct may be overestimatedbecause of associated swelling.

[0136] The analysis of variance for repeated measures was used toanalyze the hematocrit, glucose, ABG, rCBF, and ICP in the control andtreated groups. If significance was noted, the values of these variablesimmediately before the t-PA and/or MHM23 administration were thencompared by the Student's t test. When necessary, the analysis ofcovariance was used to compare the control and treated groups. After asignificant treatment-by-time interaction, individual contrasts wereused to compare the treatment means at each time point; that is, if asignificant treatment-by-time interaction was noted, the treatmenteffects were examined at each time point. The infarct size and specificgravities of the brain were compared (treated versus control) by theStudent's t test. All the results were two-sided and were evaluated byusing α=0.05.

Results

[0137] The results of the above experiment are depicted in FIGS. 1-3. Asshown in FIGS. 1 and 2, administration of anti-CD18 antibody alone, leadto a significant increase in cerebral blood flow as well as asignificant reduction in infarct size relative to control. FIG. 3 showsthat anti-CD18 antibody alone, t-PA alone, or a combination of these twoagents tend to reduce intracranial pressure (ICP) at 6-7 hours.Furthermore, the experiments show that anti-CD18 antibody is compatablewith t-PA and improves the outcome of t-PA when given at 3-5 hours afterinstallation of the clot into the cerebral circulation.

[0138] The increase in cerebral blood flow and the reduction in infarctsize observed in the above experiments are thought to be predictive ofan improvement in clinical outcome as measured by a standard strokescale. Accordingly, this application provides a method for improvingclinical outcome in patients having suffered stroke as defined herein.

[0139] The model described in this example differs from that previouslydescribed in Bednar et al., Stroke 23(1):152 (1992) in that the animalsin the study were not subjected to extraneous systemic hypotension (byreducing the mean arterial pressure in the animal to 30 mmHg bycontrolled exsanguination). Also, the anti-CD18 antibody was given morethan 30 minutes after the thromboembolic event and the dose wasdifferent.

1 15 98 amino acids Amino Acid Linear 1 Ala Ser Thr Lys Gly Pro Ser ValPhe Pro Leu Ala Pro Ser Ser 1 5 10 15 Lys Ser Thr Ser Gly Gly Thr AlaAla Leu Gly Cys Leu Val Lys 20 25 30 Asp Tyr Phe Pro Glu Pro Val Thr ValSer Trp Asn Ser Gly Ala 35 40 45 Leu Thr Ser Gly Val His Thr Phe Pro AlaVal Leu Gln Ser Ser 50 55 60 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr ValPro Ser Ser Ser 65 70 75 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn HisLys Pro Ser 80 85 90 Asn Thr Lys Val Asp Lys Arg Val 95 98 amino acidsAmino Acid Linear 2 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala ProCys Ser 1 5 10 15 Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys LeuVal Lys 20 25 30 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser GlyAla 35 40 45 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser50 55 60 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn 6570 75 Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 80 8590 Asn Thr Lys Val Asp Lys Thr Val 95 98 amino acids Amino Acid Linear 3Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser 1 5 10 15Arg Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 20 25 30 AspTyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 35 40 45 Leu ThrSer Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 50 55 60 Gly Leu TyrSer Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 65 70 75 Leu Gly Thr GlnThr Tyr Thr Cys Asn Val Asn His Lys Pro Ser 80 85 90 Asn Thr Lys Val AspLys Arg Val 95 98 amino acids Amino Acid Linear 4 Ala Ser Thr Lys GlyPro Ser Val Phe Pro Leu Ala Pro Cys Ser 1 5 10 15 Arg Ser Thr Ser GluSer Thr Ala Ala Leu Gly Cys Leu Val Lys 20 25 30 Asp Tyr Phe Pro Glu ProVal Thr Val Ser Trp Asn Ser Gly Ala 35 40 45 Leu Thr Ser Gly Val His ThrPhe Pro Ala Val Leu Gln Ser Ser 50 55 60 Gly Leu Tyr Ser Leu Ser Ser ValVal Thr Val Pro Ser Ser Ser 65 70 75 Leu Gly Thr Lys Thr Tyr Thr Cys AsnVal Asp His Lys Pro Ser 80 85 90 Asn Thr Lys Val Asp Lys Arg Val 95 107amino acids Amino Acid Linear 5 Arg Thr Val Ala Ala Pro Ser Val Phe IlePhe Pro Pro Ser Asp 1 5 10 15 Glu Gln Leu Lys Ser Gly Thr Ala Ser ValVal Cys Leu Leu Asn 20 25 30 Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln TrpLys Val Asp Asn 35 40 45 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val ThrGlu Gln Asp 50 55 60 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu ThrLeu Ser 65 70 75 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu ValThr 80 85 90 His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly95 100 105 Glu Cys 105 amino acids Amino Acid Linear 6 Gln Pro Lys AlaAla Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu GlnAla Asn Lys Ala Thr Leu Val Cys Leu Ile Ser 20 25 30 Asp Phe Tyr Pro GlyAla Val Thr Val Ala Trp Lys Ala Asp Ser 35 40 45 Ser Pro Val Lys Ala GlyVal Glu Thr Thr Thr Pro Ser Lys Gln 50 55 60 Ser Asn Asn Lys Tyr Ala AlaSer Ser Tyr Leu Ser Leu Thr Pro 65 70 75 Glu Gln Trp Lys Ser His Arg SerTyr Ser Cys Gln Val Thr His 80 85 90 Glu Gly Ser Thr Val Glu Lys Thr ValAla Pro Thr Glu Cys Ser 95 100 105 100 amino acids Amino Acid Linear 7Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Pro 1 5 10 15Lys Asn Ser Ser Met Ile Ser Asn Thr Pro Ala Leu Gly Cys Leu 20 25 30 ValLys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 35 40 45 Gly AlaLeu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 50 55 60 Ser Ser GlyLeu Tyr Ser Leu Ser Ser Val Val Thr Val Pro His 65 70 75 Gln Ser Leu GlyThr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 80 85 90 Pro Ser Asn Thr LysVal Asp Lys Arg Val 95 100 11 amino acids Amino Acid Linear 8 Pro LysAsn Ser Ser Met Ile Ser Asn Thr Pro 1 5 10 8 amino acids Amino AcidLinear 9 His Gln Asn Leu Ser Asp Gly Lys 1 5 232 amino acids Amino AcidLinear 10 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 15 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Tyr Thr Phe Thr 2025 30 Glu Tyr Thr Met His Trp Met Arg Gln Ala Pro Gly Lys Gly Leu 35 4045 Glu Trp Val Ala Gly Ile Asn Pro Lys Asn Gly Gly Thr Ser His 50 55 60Asn Gln Arg Phe Met Asp Arg Phe Thr Ile Ser Val Asp Lys Ser 65 70 75 ThrSer Thr Ala Tyr Met Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90 Thr AlaVal Tyr Tyr Cys Ala Arg Trp Arg Gly Leu Asn Tyr Gly 95 100 105 Phe AspVal Arg Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val 110 115 120 Thr ValSer Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 125 130 135 Ala ProSer Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 140 145 150 Cys LeuVal Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 155 160 165 Asn SerGly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 170 175 180 Leu GlnSer Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 185 190 195 Pro SerSer Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 200 205 210 His LysPro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 215 220 225 Ser CysAsp Lys Thr His Thr 230 214 amino acids Amino Acid Linear 11 Asp Ile GlnMet Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp ArgVal Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn 20 25 30 Asn Tyr Leu AsnTrp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45 Leu Leu Ile Tyr TyrThr Ser Thr Leu His Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser GlySer Gly Thr Asp Tyr Thr Leu Thr Ile 65 70 75 Ser Ser Leu Gln Pro Glu AspPhe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90 Gly Asn Thr Leu Pro Pro Thr PheGly Gln Gly Thr Lys Val Glu 95 100 105 Ile Lys Arg Thr Val Ala Ala ProSer Val Phe Ile Phe Pro Pro 110 115 120 Ser Asp Glu Gln Leu Lys Ser GlyThr Ala Ser Val Val Cys Leu 125 130 135 Leu Asn Asn Phe Tyr Pro Arg GluAla Lys Val Gln Trp Lys Val 140 145 150 Asp Asn Ala Leu Gln Ser Gly AsnSer Gln Glu Ser Val Thr Glu 155 160 165 Gln Asp Ser Lys Asp Ser Thr TyrSer Leu Ser Ser Thr Leu Thr 170 175 180 Leu Ser Lys Ala Asp Tyr Glu LysHis Lys Val Tyr Ala Cys Glu 185 190 195 Val Thr His Gln Gly Leu Ser SerPro Val Thr Lys Ser Phe Asn 200 205 210 Arg Gly Glu Cys 450 amino acidsAmino Acid Linear 12 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val GlnPro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Tyr ThrPhe Thr 20 25 30 Glu Tyr Thr Met His Trp Met Arg Gln Ala Pro Gly Lys GlyLeu 35 40 45 Glu Trp Val Ala Gly Ile Asn Pro Lys Asn Gly Gly Thr Ser His50 55 60 Asn Gln Arg Phe Met Asp Arg Phe Thr Ile Ser Val Asp Lys Ser 6570 75 Thr Ser Thr Ala Tyr Met Gln Met Asn Ser Leu Arg Ala Glu Asp 80 8590 Thr Ala Val Tyr Tyr Cys Ala Arg Trp Arg Gly Leu Asn Tyr Gly 95 100105 Phe Asp Val Arg Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val 110 115120 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 125 130135 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 140 145150 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 155 160165 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 170 175180 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 185 190195 Thr Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp 200 205210 His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys 215 220225 Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly 230 235240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265270 His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Met 275 280285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 290 295300 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp 305 310315 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 320 325330 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 335 340345 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 350 355360 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 365 370375 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 380 385390 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 395 400405 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 410 415420 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 425 430435 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 440 445450 7 amino acids Amino Acid Linear 13 His Gln Ser Leu Gly Thr Gln 1 5 8amino acids Amino Acid Linear 14 His Gln Asn Ile Ser Asp Gly Lys 1 5 8amino acids Amino Acid Linear 15 Val Ile Ser Ser His Leu Gly Gln 1 5

1. A method for increasing cerebral blood flow and/or reducing infarctsize in focal ischemic stroke caused by obstruction of a main cerebralartery in a human mammal which comprises the step of co-administeringeffective amounts of tissue plasminogen activator (tPA) and anti-CD18antibody to the mammal wherein neither the tPA nor the anti-CD18antibody is administered to the mammal until about three to five hoursafter the onset of focal ischemic stroke.
 2. The method of claim 1 thatincreases cerebral blood flow and reduces infarct size in the mammal. 3.The method of claim 1 wherein the anti-CD18 antibody is an antibodyfragment.
 4. The method of claim 3 wherein the anti-CD18 antibodyfragment is a F(ab′)₂.
 5. The method of claim 1 wherein the anti-CD18antibody is humanized.
 6. The method of claim 1 wherein the anti-CD18antibody is administered to the mammal by bolus dosage.
 7. The method ofclaim 1 wherein the anti-CD18 antibody is administered inravenously. 8.The method of claim 1 wherein the anti-CD18 antibody is administered viacontinuous infusion.
 9. The method of claim 1 wherein the anti-CD18antibody and the tPA are simultaneously administered to the mammal. 10.The method of claim 1 wherein the anti-CD18 antibody is administeredbefore the tPA is administered to the mammal.
 11. The method of claim 1wherein the anti-CD18 antibody is humanized H52 antibody comprisingheavy chain sequence of SEQ ID NO:10 and light chain sequence of SEQ IDNO:11.
 12. The method of claim 11 wherein the H52 antibody is a F(ab′)₂.13. The method of claim 1, wherein the anti-CD18 antibody binds to anextracellular domain of CD18 and inhibits or reduces the ability of acell expressing CD18 to bind to endothelium.
 14. The method of claim 1,wherein the anti-CD18 antibody binds CD18 with an affinity of 4 nm orless.
 15. The method of claim 1, wherein the anti-CD18 antibody bindsCD18 with an affinity of 3 nm or less.
 16. The method of claim 1,wherein the anti-CD18 antibody binds CD18 with an affinity of 1 nm orless.
 17. The method of claim 1, wherein the anti-CD18 antibodydissociates the CD11b/CD18 complex.
 18. The method of claim 1, whereinthe anti-CD18 antibody binds to the epitope bound by H52 antibody.